About ZR Fungal/Bacterial RNA MicroPrep™
The ZR Fungal/Bacterial RNA MicroPrep™ provides for rapid isolation of total RNA from pelleted tough-to-lyse bacterial (e.g., Gram-positive), yeast, and/or fungal cells. The kit employs ultra-high density BashingBeads™ for sample homogenization and a robust buffer system for total RNA purification (small RNAs included). Using Fast-Spin column technology, the RNA is eluted into volumes as little as 6 µl and suitable for subsequent procedures including RT-PCR. The entire RNA isolation procedure typically takes less than 15 minutes.
|Processing Time||15 min|
|Equipment||Microcentrifuge, vortex and/or cell disrupter/pulverizer (optional).|
|Sample Sources||Fresh or frozen pelleted cells (bacteria, yeast, fungi).|
|RNA Purity||High quality RNA (A260/A280>1.8, A260/A230> 1.8) recovered.|
|RNA Recovery||Up to 5 µg RNA can be eluted into ≥ 6 µl, allowing for a highly concentrated sample.|
|RNA Storage||RNA is eluted with RNase-free water and can be stored at ≤ -70°C. The addition of RNase inhibitors is optional but highly recommended for prolonged storage.|
Total RNA was isolated from equal amounts of pelleted E. coli cells containing plasmid DNA (pGEM®) using the ZR Fungal/Bacterial RNA MicroPrep™ or kit from Supplier A. The samples were resolved in a 2% (w/v) agarose gel. RNA Millenium Markers (Ambion) and ZR 1 kb DNA Marker (Zymo Research) were used.
* = genomic (< 10 kb) and plasmid (< 3 kb) DNA contamination;
Resuspend a fresh or frozen cell pellet in 800 µl RNA Lysis Buffer2 (≥4 volumes)2 (e.g., 800 µl buffer will allow for processing of up to 400 µl lysate in Step 4) and transfer the mixture to a ZR BashingBead™ Lysis Tube.
Secure in a bead beater3 fitted with a 2 ml tube holder assembly and process (e.g., Disruptor Genie™ - bacterial/yeast cells: 1-2 minutes at maximum speed).
Centrifuge the ZR BashingBead™ Lysis Tube at ≥12,000 x g for 1 minute.
Transfer 400 µl supernatant to a Zymo-Spin™ IIIC Column in a Collection Tube and centrifuge at 8,000 x g for 30 seconds.
Add 0.8 volume ethanol (95-100%) to the flow-through in the Collection Tube and mix well (e.g., 320 µl ethanol added to 400 µl flow-through).
Transfer the mixture to a Zymo-Spin™ IC Column4 in a Collection Tube and centrifuge at ≥12,000 x g for 30 seconds5. Discard the flow-through.
Add 400 µl RNA Prep Buffer to the column. Centrifuge at ≥12,000 x g for 1 minute. Discard the flow-through and replace the Zymo-Spin™ IC Column back into the Collection Tube.
Add 800 µl RNA Wash Buffer to the column. Centrifuge at ≥12,000 x g for 30 seconds. Discard the flow-through and replace the Zymo-Spin™ IC Column back into the Collection Tube. Repeat the wash step with 400 µl RNA Wash Buffer.
Centrifuge the Zymo-Spin™ IC Column at ≥12,000 x g for 2 minutes in the emptied Collection Tube to ensure complete removal of the wash buffer.
Carefully remove the Zymo-Spin™ IC Column from the Collection Tube and place into a DNase/RNase-Free Tube. Add ≥6 µl DNase/RNase-Free Water directly to the column matrix and let stand for 1 minute.
Centrifuge at 10,000 × g for 30 seconds to elute the RNA from the column. RNA can be used immediately or stored at ≤-70 oC (see Specifications).